Genomic enzymology to decipher bacterial membrane-associated glycan assembly

The National Institute of General Medical Sciences awarded the Trustees of Boston University $437,036 on June 1, 2026, for genomic enzymology research to decipher bacterial membrane-associated glycan assembly under the Biomedical Research and Research Training program (CFDA 93.859).

The funded work identifies and assigns functional signatures for substrate specificity in phosphoglycosy transferase and glycosyltransferase enzymes to enable glycan composition inference at the protein sequence level. The research focuses on the monotopic phosphoglycosyl transferase superfamily, which is exclusive to prokaryotes, with the goal of developing selective inhibitors targeting bacterial survival and virulence pathways without affecting eukaryotic systems. The recipient will conduct structural and functional analysis of glycosyltransferases to identify specificity determinants for cognate substrates, building on prior work characterizing monotopic phosphoglycosyl transferase structure, dynamics, membrane topology, and sequence similarity networks.

Work is performed in Boston, Massachusetts, with a period of performance from June 1, 2026, through May 31, 2031. This is a Project Grant, the standard assistance type for investigator-initiated biomedical research under NIGMS's mission to support basic research advancing understanding of biological processes and disease diagnosis, treatment, and prevention.

Abstract

Glycoconjugates are critical for bacterial survival and virulence, as they play key roles in cell-wall integrity, immune evasion, and host-pathogen interactions. The biosynthesis of many bacterial glycoconjugates is achieved via an "EN bloc" transfer strategy. Biosynthesis is initiated by membrane-embedded polyprenol phosphate (prenp) phosphoglycosyl transferases (pgts), that catalyze the first membrane-committed step, which involves the transfer of a phosphosugar from a nucleoside diphosphate (NDP)-activated donor to an amphiphilic prenp acceptor. This step affords a membrane-bound prenpp-sugar product, which is then elongated by the action of a cadre of glycosyltransferases (GTS) that define each new glycosidic linkage to "code" the new glycan. The assembled prenpp-glycan is then transferred across the membrane by a flippase and ultimately the glycan is transferred to protein, lipid or glycan acceptors. In prokaryotes, NDP-sugar substrates display greater variation compared to eukaryotes, which significantly broadens the diversity of glycoconjugate products in microorganisms. The proposed work will to identify and assign signatures for substrate specificity in PGT and GT enzymes such that the glycan composition can ultimately be inferred at the protein sequence level. Moreover, the monotopic PGT (monopgt) superfamily is exclusive to prokaryotic, allowing for the development of selective inhibitors towards at crucial survival and virulence targets without affecting eukaryotic systems. Our recent research has shed light on the monopgt structure, dynamics, and membrane topology. We have developed a sequence similarity network with isofunctional groupings that provide sequence motifs for functional assignment across the monopgt superfamily. Analysis of the products and structures of GTS from campylobacter has allowed identification of specificity determinants for their cognate UDP-sugar substrate. Changes in sugar specificity are found to be reflected in the evolutionary divergence of members later in the pathway. Future research aims to understand enzyme specificity through genomic enzymology, focusing on pathways from pathogens. We will apply bioinformatics, biochemical analysis, and structural biology approaches to connect primary sequence information to UDP-sugar substrate specificity for the monopgt, GT-b and GT-a superfamilies. The GT structures allow the pursuit of computational studies of the dynamics of GT-substrate complexes to assess membrane topology and enzyme conformation as a specificity determinant and possible effector of processivity. Structures of the bifunctional (BI) monopgt family members combined with informatics will shed light on protein:protein interactions between pathway enzymes and provide insight into metabolon formation in glycan biosynthesis. We propose leveraging structure prediction to identify membrane-associated GT-BS to uncover new metabolic pathways for glycan biosynthesis. Ultimately, integration of computational and experimental studies will further elucidate the interactions between enzymes, membranes, and substrates, uncovering new pathways and regulatory mechanisms critical to bacterial survival and pathogenesis.